Oxidative stress and glutathione (GSH) imbalance are major contributors to the pathogenesis of diabetic nephropathy. Circulating monocytes participate to this process since these cells carry a high oxidative burden and are found in diabetic renal tissue. Current options for the treatment of oxidative stress in diabetic nephropathy are limited and only partially effective, thus interest in the development of new strategies is high. N-acetylcysteine (NAC) and the milk thistle (MTh) plant flavonolignans are nutritional supplements with complementary antioxidant properties. Both supplements are capable of neutralizing directly toxic free radicals but, more importantly, NAC is substrate for the intracellular generation of GSH and the MTh flavonolignans are inducers of many cellular enzymes participating in GSH metabolism, including GSH-reductase (GSH-R), GSH-peroxidase (GSH-Px), GSH-S-transferases (GST) and superoxide dismutase (SOD). We propose that combined oral supplementation of NAC and MTh flavonolignans will reduce proteinuria and urinary and systemic manifestations of oxidative stress and inflammation, which are characteristically observed in patients with T2DM and related nephropathy. We expect these effects to be achieved with minimal or no side effects, and with good patient tolerance. To test this hypothesis, we propose a double-blind randomized, placebo-controlled, five-arm pilot study which includes a dose ranging component in patients with T2DM and established nephropathy. Intervention will consist of the individual and combined oral administration of one level of NAC and two levels of MTh flavonolignans or placebo for three months. The primary outcome measure will be urinary excretion of albumin, a marker of glomerular injury. Secondary outcome measures will be alpha-1 microglobulin, a marker of tubular injury, and urinary excretion of inflammatory cytokines and C-C chemokines, i.e. markers of renal inflammation. In plasma and in peripheral blood monocytes from the same patients, we will analyze GSH content and activity of GSH metabolizing enzymes. In addition, we will analyze the plasma and urine glycoproteome, with focus on those glycoproteins serving as inflammatory cell messengers and hormones. These variables will be monitored in relation to both treatment allocation and prevalent blood and urine levels of the active treatment. Throughout the trial, we will monitor the safety and tolerability of this combination treatment. Validation of the proposed hypothesis will provide preliminary evidence of efficacy, safety and tolerability of combination treatment with NAC and MTh flavonolignans and it will be the necessary first step for the future design of more definitive clinical investigation and of mechanistic research in animal models of T2DM and diabetic nephropathy. PUBLIC HEALTH RELEVANCE: Oxidative stress (OS) consists of high production and low disposal of toxic pro-oxidant chemicals, and it is very common in patients with Type 2 Diabetes Mellitus (T2DM) and diabetic kidney disease. Veterans have high prevalence of T2DM, diabetic kidney disease, and associated OS. OS causes progression of diabetic kidney disease, with spilling of protein in the urine and, eventually, with the need for dialysis or kidney transplantation. In most cases, the available treatments cannot stop the progression of diabetic kidney disease and the spilling of protein in the urine. We suspect this is because no conventional treatments can completely correct OS in diabetic kidney disease. We propose to test if the combination of two potent antioxidant nutritional supplements, N-acetylcysteine and the milk thistle extract silymarin, is capable of correcting the OS and the spilling of protein in the urine in Veterans with T2DM and diabetic kidney disease.